Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity

Bacterial glycomes are rich in prokaryote-specific or “rare” sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. In vitro, RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.


A)
B) Estimated percent conversion* of S-1P to NDP-sugar.  Figure S1. Time course of coupling reactions of Glc-1P with dTMP-Imidazolide to evaluate different catalysts. (a) Representative 31 P-NMR analyses of coupling reactions using α-Glc-1P and dTMP-Imidazolide (dTMP-Im) substrates with different catalysts indicates NMI-HCl is the best of tested catalysts at promoting formation of the desired product (t = 18 hr) (PP = pyrophosphate). Full consumption of dTMP-Im (~ -9 ppm) to the desired asymmetric pyrophosphate bond was indicated by the appearance of two doublets (~ -11 to -14 ppm). Notably, addition of 4-dimethylaminopyridine (DMAP, trace J) led to the formation of a singlet (~ -12 ppm), which represents a symmetric pyrophosphate resulting from selfdimerization of dTMP, as confirmed by mass spectrometry. 3 See table in part B for abbreviations of catalysts tested. Catalysts were chosen based on a previous study on the optimization of phosphate condensation for glycolipid synthesis. 1 (B) Quantification of percent conversion to NDP-sugars using indicated catalysts as monitored by 31 P-NMR over a long time course (for t = 18 hr, traces shown in part A). *Calculated as shown in Figure S1B. (A) Representative 31 P-NMR analyses of coupling reactions using β-L-Rha-1P minus (left) or plus NMI-HCl (right) using the same reagent ratios as Figure S1A over the indicated time periods demonstrate that NMI-HCl accelerates the reaction, and it is nearly complete in t = 3 hr with catalyst added. Note that 20% additional dTMP-Im was added to the reaction with catalyst at t = 3 hr. (B) Quantification of percent conversion to NDP-sugar based on data shown in part A.   , and 0.008 mg/mL of PPiase in the absence or presence of indicated fragments (100 µM) of dTDP-β-L-Rha, and percent dTDP-α-Glc produced was measured (t = 6 min, 25 °C). Bars indicate SD (n = 2). Figure S8. Mutation of E256 in SALTY RmlA leads to a competitive inhibition mechanism by dTDP-β-L-Rha (1). Indicated reactions were set-up in the presence and absence of dTDP-β-L-Rha at concentrations that led to measurable inhibition for each SALTY RmlA construct. While D104N (stabilizing mutation) and D104N/Y146F (active site mutation) experience mixed inhibition by dTDP-β-L-Rha, D104N/E256D (allosteric site mutation) is competitively inhibited by dTDP-β-L-Rha, demonstrating that allosteric regulation is lost upon mutation of E256. Briefly, Glc-1P was fixed at 100 μΜ and dTTP was titrated. For each reaction, 12.5 nM of RmlA enzyme and 0.008 mg/mL PPiase was pre-incubated at 25 °C in buffer (35 mM Tris-HCl pH 7.5, 2.2 mM MgCl2) +/-dTDP-β-L-Rha prior to addition of substrates. Bars indicate standard error of the mean (SEM, n = 3).   Overlaid HPLC analyses of indicated reactions demonstrates that activation of the non-canonical substrate, β-L-Man-1P using dTTP nears completion using RmlA* but not RmlA D104N. Note that reaction products were treated with shrimp alkaline phosphatase (rSAP) to confirm production of product and aid in quantification. (D) Quantification of dTDP-sugar produced from part C. (E) Quantification of dTDP-β-L-Man produced with indicated RmlA mutants, including an alternative allosteric site mutation (H117A), indicates RmlA* still shows the highest product production (performed over a shorter reaction time than parts A and C). Reactions were carried out in a final volume of 30 μL containing 5 μM of indicated RmlA protein, 0.008 mg/mL PPiase, 2 mM dTTP, and 10 mM S-1P (except 2 mM S-1P used in part E) in buffer (100 mM MOPS, pH 7.5, and 7.5 mM MgCl2, 37 °C, t = 24 hr for all but part E, for which t = 6 hr). For part C, 1 μL of rSAP (33.33 U/mL) was subsequently added to each reaction (after t = 24 hr) and incubated for t = 2 hr at 37 °C.   Figure 1B). See Table S1 for a comparison of predicted energies of each conformation. *Calculation was performed on a cluster computer using Spartan'20 Software. An initial conformational search with Molecular Mechanics (MMFF, ≤ 160 kJ/mol) of a maximal 500 potential conformers produced the lowest energy 1 C4 and 4 C1 chair conformations, which were then minimized (B3LYP/6-31G*) giving the ground-state free energies. 4 The ΔGa is the free energy gap between 1 C4 conformer and 4 C1 conformer of each anomer (

A) B) Estimated percent conversion* from S-1P to NDP-sugar
, which showed that the 1 C4 conformer is thermodynamically more stable among the calculated molecules regardless of the configuration of the anomeric position. ΔGb is the free energy gap between the 1 C4 conformers of the β-anomer and α-anomer (Eβ[ 1 C4]-Eα[ 1 C4]). The β-anomer of the free S-1P is predicted to be more stable than the α-anomer. using Schrodinger Glide with docking scores shown. β-L-Man-1P (in the lower energy 1 C4 conformation) is shown in two low energy poses that are similar to the docked α-Glc-1P, which suggests another conformation in which the substrates may bind prior to reaction. The nucleophilic O and electrophilic P of the enzymatic reaction are indicated, along with homologous SALTY residues to those highlighted in Pa RmlA in part A. Molecular docking was performed using Glide (Schrödinger Release 2021-2) 5 loaded with a crystal structure of SALTY RmlA complexed with dTTP. Default parameters were used for optimization, and an OPLS3e force field was employed. The Glide docking grid of the receptor was generated to include the dTTP and predicted S-1P binding pockets. Default van der Waals radii parameters were used. 3D structure of β-L-Man-1P was generated following LigandPrep Wizard from the structure of RmlA-bound α-Glc-1P in a published crystal structure (PDB ID: 1G23). The indicated scores are GlideScores, which approximate the free binding energies of each ligand.

General experimental methods for synthetic compounds 1.1 Ion exchange for isolation of triethylammonium (TEA) salt of nucleotides and S-1Ps
Commercially purchased nucleoside-monophosphates (dTMP, UMP and GMP) and Glc-1P were dissolved in water (0.1-0.2 M). The solution was shaken with 2.5 equivalents of IR-120 H + type ion exchange resin at room temperature for 1 hour. The solution was filtered, and the resin was washed with three times the resin volume with water. The combined filtrate was cooled in an ice-bath and two equivalents of TEA was slowly added into the shaking flask. The neutralized solution was lyophilized, and the number of counter-ions was determined by 1 H NMR. 1.2 Chemical activation of (d)NMPs NMP triethylammonium salt was dissolved in dry DMF (4 mL/mmol), and 2.5 equivalents of carbonyldiimidazole (CDI) was added. The reaction was stirred at room temperature until no NMP was observed on ESI-MS. Dry methanol (0.1 mL/mmol (d)NMP) was added in an ice-bath, and the mixture was stirred for another 30 minutes to quench the excess CDI. The reaction solution was poured into 100 mM NaClO4 in acetone-ether (v/v = 1:1). The resulting suspension was stirred for another 10 minutes and centrifuged (4 ºC, 1857 x g, 5 min). The precipitation was collected, washed with cold ether, and centrifuged again. The crude (d)NMP-imidazolide (dNMP-Im) was obtained as a white/pale yellow solid after drying under a vacuum. The successful activation of NMP was confirmed by 31 P NMR analysis, with product formation indicated by a ~-8.6 ppm signal and no monophosphate signal. Synthesis of compound 6a 3 g of L-rhamnose was stirred with 893 mg (0.4 eq) of DMAP in 25 mL of pyridine (Py) at 0 ℃. 17.3 mL (10 eq) of acetic anhydride (Ac2O) was added in portions over 30 minutes. The reaction was warmed to room temperature and kept stirring for 18 hours. The reaction mixture was then poured into 100 mL of icecooled water, and extracted with 150 mL of ethyl acetate (EA). The organic layer was washed with 50 mL of 1M HCl, 50 mL of water, 50 mL of sat. NaHCO3 (aq.), and 50 mL of brine, then dried over Na2SO4. The crude product was obtained after filtration and evaporation, and directly used in the next step without further purification.
The crude product of acetylation was stirred with 3 mL (1.5 eq) of benzylamine (BnNH2) in 25 mL of dry tetrahydrofuran (THF) at room temperature overnight. The solvent was removed. The residue was dissolved with EA, washed with water and sat. NaHCO3 (aq.), and dried over Na2SO4. The product was obtained after silica gel flash chromatography (Hex/EA = 5:1 to 3:1 to 1:1) with a yield of 62% over two steps and a weight of 3.27 g of pale-yellow oil as the final product. Compound 9a-α 103 mg of compound 9a-α was stirred with 28 mg of PtO2 (0.6 eq) in 2 mL of ethyl acetate/2 mL of ethanol under 1.1 atm of H2 overnight. The catalyst was filtered out and the solvent was concentrated to dryness. The crude product was dissolved in a mixture of 1 mL of TEA/2 mL of water/2 mL of methanol and stirred at 55 ℃ over 36 hours. The solvent was removed, and the residue was dissolved in 10 mL of water, and washed 3 times with 3 mL of DCM. The aqueous layer was lyophilized, giving 75 mg of pale-yellow oil as product 3-α (L-Rha-1P •nTEA, n = 2 determined by 1 H NMR) with a yield of 84%.

Synthesis of compound 6b
1 g of L-mannose was stirred with 366 mg (0.5 eq) of DMAP in 10 mL of Py at 0 ℃. 7 mL (12.5 eq) of Ac2O was added in portions over 30 minutes. The reaction was warmed to room temperature and kept stirring for 18 hours. The reaction mixture was poured into 40 mL of ice-cooled water, and then extracted with 80 mL of EA. The organic layer was washed with 30 mL of 1M HCl, 30 mL of water, 30 mL of sat. NaHCO3 (aq.), and 30 mL of brine, then dried over Na2SO4. The crude product was obtained after filtration and evaporation, and directly used in the next step without further purification.
The crude product of acetylation was stirred with 1 mL (1.5 eq) of BnNH2 in 10 mL of dry THF at room temperature overnight. The solvent was removed. The residue was dissolved in EA, washed with water and sat. NaHCO3 (aq.), then dried over Na2SO4. The product was obtained after silica gel flash chromatography (Hex/EA = 5:1 to 3:1 to 1:1) as 1.62 g of pale-yellow oil as product with a yield of 81%. Synthesis of compound 9b 1.60 g of 6b and 1.12 g (2 eq) of DMAP were stirred in 20 mL of dry DCM at room temperature. 1.9 mL (2 eq) of diphenyl phosphoryl chloride in 6 mL of dry DCM was added into the reaction mixture dropwise over a period of 1 hour. The reaction solution was stirred for another 2 hours. The reaction was diluted with 50 mL of DCM, washed with 40 mL of NaHCO3 (sat. aq.), 40 mL of 0.1 M HCl, and then dried over Na2SO4, filtered, and concentrated. The product was purified via silica gel flash chromatography (Hex:EA = 3:1 to 3:2 to 2:3) and to give 1.72 g of pale yellow oil as the major product 9b (β-phosphate, 64%) and 0.60 g of pale yellow oil as the minor product 9b-α (α-anomer, 22%). Compound

Synthesis of 6-deoxy-β-L-talose-1-phosphate (5)
This is the first reported synthesis of β-L-6dTal-1P (5). The synthesis of precursor 8 was performed following a protocol reported by Danieli et. al. 10   Synthesis of compound S1 3.3 mL (4 eq) of dry DMSO was added dropwise into a stirring solution of 2 mL (2 eq) of oxalyl chloride in 15 mL of dry DCM at -78 ℃. A solution of 2.87 g of compound 7 in 30 mL of dry DCM was added into the stirring solution over a 1 hour period at -78 ℃. After the addition of 8.2 mL (4 eq) of DIPEA, the reaction mixture was warmed to room temperature and stirred for another hour until TLC analysis indicated that the reaction was complete. The mixture was quenched by the addition of an equal volume of sat.
NaHCO3 and extracted with 50 mL of DCM. The organic layer was dried over Na2SO4 and concentrated to dryness, giving the crude product that could be used directly in the next step.

Synthesis of compound 8
Crude S1 was stirred in 40 mL of ethanol at 0 ℃. 450 mg (1 eq) of NaBH4 was added and the reaction was stirred at 0 ℃ for another hour. The reaction was quenched by the addition of 20 mL of brine. The resulting mixture was extracted with EA (3 x 50 mL). The combined organic layers were dried over Na2SO4, concentrated to dryness, and purified via silica gel flash chromatography (Hex:EA = 100:0 to 9:1 to 4:1), giving 2.26 g of colorless oil as product 8 with a yield of 79%.

Synthesis of compound S2
2.26 g of 8 in 10% acetic acid (AcOH,100 ml) was stirred at 50 ℃ for 5 hour, and monitored by TLC (1:1 Hex/EA). The mixture was co-evaporated with toluene, then the residue was dissolved in 30 mL of dry Py. 678 mg (0.6 eq) of DMAP and 5.3 mL (6 eq) of Ac2O was added, and the solution was stirred overnight at room temperature. The reaction mixture was poured into 60 mL of ice-cooled water, and extracted with 2 x 100 mL of EA. The organic layer was washed with 50 mL of 1 M HCl, 50 mL of water, 50 mL of sat. NaHCO3 (aq), and 50 mL of brine, then dried over Na2SO4. The crude product after filtration and evaporation was purified via silica gel flash chromatography (Hex:EA = 100:0 to 8:1 to 7:2) to give 2.71 g of pale-yellow oil as product with a yield of 89%. 188 mg of compound 9c was stirred with 52 mg of PtO2 in 2 mL of EA/2 mL of ethanol under 1.1 atm of H2 overnight. The catalyst was filtered out and the solvent was concentrated to dryness. The crude product was dissolved in a mixture of 1 mL TEA/2 mL of water/2 mL of methanol and stirred at 55 ℃ over 36 hours. The solvent was removed, and the resulting residue was dissolved with 10 mL of water, and then washed 3 times with 3 mL of DCM. The aqueous layer was lyophilized, giving 108 mg of pale-yellow oil as product 5 (β-L-6dTal-1P•nTEA, n = 1.5 determined by 1 H NMR) with a yield of 76%.   Table  S3. PCR insertion products were DpnI treated prior to PCR purification (Qiagen), and transformation into Mach1 competent cells (Invitrogen). Following sequence confirmation by DNA sequencing (Genewiz), plasmids (Table S2) were transformed into indicated competent cells (Table S4) for overexpression.
For C-term His6-tagged M. tuberculosis RmlA purification, pellets from 500 mL of culture were each resuspended on ice with 25 mL of lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10% glycerol, 20 mM imidazole). Cells were rocked for 30 min at 4 °C and were sonicated (Fisherbrand, 5 min, 30 sec on/30 sec off, 50% amplitude, twice with cooling between runs). Cellular debris was cleared by centrifugation (Beckman Coulter Allegra X-15R at 10,956 x g for 30 min at 4 °C ). For each, supernatant was added to 3 mL of pre-washed/equilibrated Ni-NTA resin (Qiagen) and rocked for 1 hr at 4 °C. The column was washed with 2 column volumes (CVs) of 25 mM imidazole in lysis buffer. The targeted His-tagged proteins were eluted with 1 CV of elution buffer (250 mM imidazole in lysis buffer). Eluted fractions were concentrated using an Amicon Ultra Centrifugal Filter Device (Millipore, 10 kD molecular weight cut off (MWCO)) by centrifugation (1857 x g, 4 °C) and buffer exchanged into 10 mM tris(hydroxylmethyl)aminomethane hydrochloride (Tris-HCl), pH 8.0, 20 mM NaCl, and 10 % glycerol.
All protein purifications were performed at 4 °C. The concentration of each purified protein was determined by the DC protein assay (Bio-Rad) using bovine serum albumin (BSA) as a standard. Proteins were aliquoted, flash frozen with N2(l), and stored at -80 °C.
Analysis bacterial RmlA inhibition by synthetic NDP-sugars. Activities of RmlA from different species were measured using a reported coupled malachite green assay. 8,13 Reactions were conducted with 12.5 nM SALTY, E.coli and P.aeruginosa RmlA and 25 nM of M. tuberculosis RmlA with 0.008 mg/mL inorganic pyrophosphatase (PPiase) in reaction buffer (35 mM Tris-HCl, pH 7.5, 2.2 mM MgCl2) 8,14 with 100 μM each dTTP and α-Glc-1P at 25 °C in a final volume of 30 μL. The linear phase was determined as shown in Figure S6D.
For each dilution series, the compounds were diluted in water and added to reactions with final concentrations of: 1000 μM, 500 μM, 250 μM, 175 μM, 100 μM, 75 μM, 50 μM, 25 μM, 10 μM, 2.5 μM, 1 μM, and 0 μM (control reaction with no inhibitors added; water was used). In a typical reaction, RmlA and PPiase were pre-incubated in reaction buffer at 25 °C for 5 min with and without inhibitors, enzymatic reactions were then initiated with the addition of 100 μM substrates, yielding a final volume of 30 μL. At t = 6 min post-initiation of each reaction, 30 μL of 0.05% formic acid was added to quench the reaction. For each set of reactions, reactions that only contained substrates and inhibitors at each concentration (12 total) in reaction buffer were prepared and treated the same as the reaction described above except no enzyme mM dTTP, and 2 mM S-1P in buffer (100 mM 3-(N-morpholino)-propanesulfonic acid (MOPS), pH 7.5, and 7.5 mM MgCl 2 ) based on conditions from a previous report. 15 Reactions were incubated at 37 °C for 6 hr (or indicated time points) and quenched by adding 30 μL of 0.05% formic acid. For analysis of synthetic S-1P activation at high concentrations, 10 mM S-1P was used and reactions were incubated at 37 °C for 24 hr. Subsequently, 1 U of 1 U/μL Shrimp Alkaline Phosphatase (rSAP) was added to the reaction, which was then incubated at 37 °C for 2 hr prior to quenching with 30 μL of 0.05% formic acid. Analytical HPLC analysis was performed on a Thermo Scientific Dionex UltiMate 3000 UHPLC+ with a Phenomenex 5 μm, 4.6 x 150 mm, NX-C18, 110 Å Gemini column. Each sample (15 μL) was injected and eluted using 50 mM triethylammonium bicarbonate (TEAB, Buffer A) and acetonitrile (Buffer B) over a linear gradient of 0-5% Buffer B or 0-10% Buffer B at a flow rate of 1.0 mL/min for t = 16 min using 254 nm wavelength for detection. To obtain the percent NDP-sugar, the area of peak representing NDP-sugar was normalized to the total area of peaks containing nucleotides (NTP, NDP, NMP, and NDP-sugar) or nucleoside if rSAP was used, which was analyzed with Chromeleon software (Thermo Scientific).

LCMS or HRMS analysis of RmlA reactions containing S-1P and (d)NTPs.
Reactions were carried out under the reaction conditions mentioned above. For LCMS analysis, quenched reactions (50 μL) were loaded into 96-well noncoated polypropylene microplates (Thermo Scientific). Analysis of standard NDPsugars and RmlA enzymatic reactions was performed on a Thermo Scientific LCMS-TQ Fortis system with an electrospray (ESI) ionization source equipped with an autosampler through a Phenomenex 5 μm, 4.6 ´ 150 mm, NX-C18, 110 Å Gemini column. Each sample (15 μL) was injected and eluted using 50 mM TEAB buffer (Buffer A) and acetonitrile (Buffer B) using a linear gradient 0-5% Buffer B at a flow rate of 0.5 mL/min for t = 16 min with detection at 254 nm. For extracted ion chromatography (EIC) analysis, SIM Q1 was employed with a selected center mass (547, 549, and 563 m/z) at a scan rate of 250 Da/sec and a scan width of 10 m/z in the negative ion mode. For general mass detection of compounds during LC analysis, full Scan Q1 was used with scan range from 250 to 1000 m/z at a scan rate of 1000 Da/sec in the negative ion mode.
For HRMS analysis of select NDP-sugars produced via chemoenzymatic reactions, HPLC fractions representing indicated peaks were lyophilized, resuspended in HPLC-grade water and analyzed by using an Agilent 6224 Accurate-Mass time-of-flight LC/MS as previously described. 8